WO2006002162A2

WO2006002162A2 - Soybean genes for resistance to aphis glycines

Info

Publication number: WO2006002162A2
Application number: PCT/US2005/021922
Authority: WO
Inventors: Curtis B. Hill; Glen L. Hartman; Yan Li; Brian Diers; Shawn Carlson
Original assignee: The Board Of Trustees Of The University Of Illinois; United States Of America, As Represented By The Secretary Of Agriculture
Priority date: 2004-06-21
Filing date: 2005-06-21
Publication date: 2006-01-05
Also published as: US8680364B2; US20110277177A1; US20060015964A1; CA2589574A1; US7994389B2; WO2006002162A3; CA2589574C

Abstract

Aphis glycines resistance (RAG) genes are provided by this invention, along with methods for identifying their presence using marker-assisted selection. Varieties of G. max and G. soja having resistance to A. glycines have been identified. The RAG genes, as well as the methods, aphid-resistant varieties, and markers disclosed herein may be used to breed new elite lines expressing soybean aphid resistance.

Description

SOYBEAN GENES FOR RESISTANCE TO APHIS GLYCINES

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No. 60/581 ,501 filed June 21 , 2004, which is incorporated by reference herein to the extent not inconsistent herewith.

BACKGROUND OF THE INVENTION [0002] This invention relates to a soybean gene for resistance to Aphis glycines, to soybean plants possessing this gene, which maps to a novel chromosomal locus, and to methods for identifying and breeding these plants, the methods involving marker-assisted selection.

[0003] Soybeans (Glycine max L. Merr.) are a major cash crop and investment commodity in North America and elsewhere. Soybean oil is one of the most widely used edible oils, and soybeans are used worldwide both in animal feed and in human food production.

[0004] A native of Asia, the soybean aphid was first found in the Midwest in 2000 (Hartman, G. L. et al., Occurrence and distribution of Aphis glycines on soybeans in Illinois in 2000 and its potential control," (1 Feb. 2001 available at a website address beginning with the usual http and www prefixes, followed by plantmanagementnetwork.org/php/default, followed by the suffix .asp.) It rapidly spread throughout the region and into other parts of North America (Patterson, J. and Ragsdale, D., "Assessing and managing risk from soybean aphids in the North Central States," (11 April 2002) available at a website address beginning with the usual http and www prefixes, followed by planthealth.info/soyaphid/aphid02, followed by the suffix .htm.) High aphid populations can reduce crop production directly when their feeding causes severe damage such as stunting, leaf distortion, and reduced pod set (Sun, Z. et al., "Study on the uses of aphid-resistant character in wild soybean. I. Aphid-resistance performance of F₂ generation from crosses between cultivated and wild soybeans," (1990) Soybean Genet, News. 17:43-48). Yield losses attributed to the aphid in some fields in Minnesota during 2001 , where several thousand aphids occurred on individual soybean plants, were >50% (Ostlie, K., "Managing soybean aphid," (2 Oct. 2002) available at a website having an address beginning with the usual http and www, followed by soybeans. umn.edu/crop/insects/aphid/aphid_publication_managingsba, and having a suffix .htm) with an average loss of 101 to 202 kg ha^"1 in those fields (Patterson and Ragsdale, supra). In earlier reports from China, soybean yields were reduced up to 52% when there was an average of about 220 aphids per plant (Wang, X.B. et al., "A study on the damage and economic threshold of the soybean aphid at the seedling stage," (1994) Plant Prot. (China) 20:12-13) and plant height was decreased by about 210 mm after severe aphid infestation (Wang, X.B. et al., "Study on the effects of the population dynamics of soybean aphid (Aphis glycines) on both growth and yield of soybean," (1996) Soybean ScL 15:243-247). An additional threat posed by the aphid is its ability to transmit certain plant viruses to soybean such as Alfalfa mosaic virus, Soybean dwarf virus, and Soybean mosaic virus (Sama, S. et al., "Varietal screening for resistance to the aphid, Aphis glycines, in soybean," (1974) Research Reports 1968-1974, pp. 171-172; Iwaki, M. et al., "A persistent aphid borne virus of soybean, Indonesian Soybean dwarf virus transmitted by Aphis glycines," (1980) Plant Dis. 64:1027-1030; Hartman, G. L. et al., supra; Hill, J. H. et al., "First report of transmission of Soybean mosaic virus and Alfalfa mosaic virus by Aphis glycines (Homoptera, Aphididae)," (1996) Appl. Entomol. Zool. 31 :178-180; Clark, A.J. and Perry, K.L., "Transmissibility of field isolates of soybean viruses by Aphis glycines," (2002) Plant Dis. 86:1219-1222).

[0005] Because A. glycines is a recent pest in the USA, a comprehensive integrated management approach to control the aphid has yet to be developed. Research to evaluate the efficacy of currently-available insecticides and other control measures has just begun.

[0006] An integral component of an integrated pest management (IPM) program to control aphids is plant resistance (Auclair, J. L., "Host plant resistance," pp. 225- 265 In P. Harrewijn (ed.) Aphids: Their biology, natural enemies, and control. Vol. C, Elsevier, New York (1989); Harrewijn, P. and Minks, A.K., "Integrated aphid management: General aspects," pp. 267-272, In A.K. Minks and P. Harrewijn (ed.) Aphids: Their biology, natural enemies, and control, Vol. C₁ Elsevier, New York (1989). Insect resistance can significantly reduce input costs for producers (Luginbill, J. P., "Developing resistant plants - The ideal method of controlling insects," (1969) USDA, ARS. Prod. Res. Rep. 111 , USGPO, Washington, D.C. Resistance was reported in G. soja (Sun, Z. et al., "Study on the uses of aphid- resistant character in wild soybean. I. Aphid-resistance performance of F₂ generation from crosses between cultivated and wild soybeans," (1990) Soybean Genet. News 17:43-48), a close relative of G. max (Hymowitz, T., "On the domestication of the soybean," (1970) Econ. Bot. 24:408-421), and other wild relatives (Zhuang, B. et al., "A study on resistance to soybean mosaic virus and Aphis glycines of perennial wild soybean," (1996) Soybean Genet. Newsl. 23:66- 69). There are no reports of resistance in G. max. A report from Indonesia indicated that there was no resistance in a test of 201 soybean cultivars and breeding lines (Sama, S. et al. (1974) Research Reports 1968-1974, p. 171-172. In Varietal screening for resistance to the aphid, Aphis glycines, in soybean. Agricultural Cooperation, Indonesia, the Netherlands).

[0007] There are numerous examples of the discovery and use of resistance genes to control aphids in crops other than soybean. Examples include Russian wheat aphid (Du Toit, F. (1987), "Resistance in wheat (Triticυm aestivum) to Diuraphis noxia (Homoptera: Aphididae)," Cereal Res. Commun. 15:175-179; wheat greenbug (Tyler, J. M., et al. (1985), "Biotype E greenbug resistance in wheat streak mosaic virus-resistant wheat germplasm lines," Crop Science25:686-688), potato aphid on tomato (Kaloshian, I., et al. (1997), "The impact of Meu-1 -mediated resistance in tomato on longevity, fecundity and behavior of the potato aphid," Macrosiphum euphorbiae," Entomol. Exp. Appl. 83:181-187), and cotton-melon aphid on melon (Klinger, J. et al. (2001), "Mapping of cotton-melon aphid resistance in melon," J. Am. Soc. Hortic. Ci. 136:56-63)

[0008] A number of soybean markers have been mapped and linkage groups created, as described in Cregan, P. B., et al., "An Integrated Genetic Linkage Map of the Soybean Genome" (1999) Crop Science 39:1464-1490. [0009] All publications referred to herein are incorporated herein by reference to the extent not inconsistent herewith.

[0010] Methods and molecular tools are needed to allow breeding of A. glycines resistance into high-yielding G. max soybean varieties.

SUMMARY OF THE INVENTION [0011] A novel method is provided for determining the presence or absence in a soybean germplasm of a gene for resistance to the soybean aphid, Aphis glycines. The aphid resistance trait has been found to be closely linked to a number of molecular markers that map to linkage group M. Two genes conferring the resistance trait are designated "R_ag1" and "R_ag2" pending approval of the Soybean Genetics Committee. The R_ag1 gene was originally discovered in the resistance source Dowling (PI548663), and the R_ag2 gene was originally discovered in the resistance source Jackson (PI548657). ("Pl" stands for "plant introductions" and these Pl numbers refer to USDA depository accession numbers.) The trait of resistance to Aphis glycines is also found in other varieties as described hereafter.

[0012] In accordance with the present invention, the gene for resistance to Aphis glycines (the RAG gene) co-segregates with molecular markers with which it is linked on linkage group M, most preferably, Satt435, Satt463, Satt245, and DOP_H14. RAG genes, exemplified by the R_ag1 and R_ag2 genes, have been found to map to a locus that lies between the markers Satt435 and Satt463. Other markers of linkage group M may also be used to identify the presence or absence of the gene. Preferably flanking markers are used for identifying the presence of a RAG gene or for marker-assisted breeding. Most preferably, the markers used map within about 20 cM, and more preferably within about 10 cM of a RAG locus (which contains one of the RAG genes), or within about 20 cM and more preferably within about 10 cM of Satt435 or Satt463.

[0013] The information disclosed herein regarding RAG loci is used to aid in the selection of breeding plants, lines and populations containing Aphis glycines resistance for use in introgression of this trait into elite soybean germplasm, or germplasm of proven genetic superiority suitable for variety release. [0014] Also provided is a method for introgressing a soybean Aphis glycines resistance gene into non-resistant soybean germplasm or less resistant soybean germplasm. According to the method, nucleic acid markers linked to a RAG gene are used to select soybean plants containing a RAG locus. Plants so selected have a high probability of expressing the trait Aphis glycines resistance. Plants so selected can be used in a soybean breeding program. Through the process of introgression, the RAG locus is introduced from plants identified using marker- assisted selection to other plants. According to the method, agronomically desirable plants and seeds can be produced containing the RAG locus from germplasm containing a RAG gene.

[0015] Particular examples of sources of R_ag1 resistance to A. glycines are the following G. max varieties: Dowling (PI548663) and its grandparent CNS (PI548445) and particular examples of sources of R_ag2 resistance to A. glycines are the following G. max varieties: Jackson (PI548657), and its parent Palmetto (PI548480). PI071506 is also a source of A. glycines resistance.

[0016] Other sources of A. glycines resistance are disclosed below.

[0017] Also provided herein is a method for producing an inbred soybean plant adapted for conferring, in hybrid combination, Aphis glycines resistance. First, donor soybean plants for a parental line containing a RAG gene, preferably R_ag1 or R_ag2, are selected. According to the method, selection can be accomplished via nucleic acid marker-associated selection as explained herein. Selected plant material may represent, among others, an inbred line, a hybrid, a heterogeneous population of soybean plants, or simply an individual plant. According to techniques well known in the art of plant breeding, this donor parental line is crossed with a second parental line. Preferably, the second parental line is high yielding. This cross produces a segregating plant population composed of genetically heterogeneous plants. Plants of the segregating plant population are screened for the RAG locus. Those plants having the RAG locus are selected for further breeding until a line is obtained that is homozygous for resistance to Aphis glycines at the RAG locus. This further breeding may include, among other techniques, additional crosses with other lines, hybrids, backcrossing, or self-crossing. The result is an inbred line of soybean plants that are resistant to Aphis glycines and also have other desirable traits from one or more other inbred lines.

[0018] Soybean plants, seeds, tissue cultures, variants and mutants having Aphis glycines resistance produced by the foregoing methods are also provided in this invention.

BRIEF DESCRIPTION OF THE DRAWINGS [0019] Figure 1 shows a published soybean genetic linkage M composite map and anchored markers, in which SSR markers used to create the linkage map with the R_ag1and R_ag2 genes are indicated by horizontal lines. The map has been broken into four consecutive vertical sections, Figures 1A through Figures 1 D.

[0020] Figure 2 is a linkage map of soybean linkage group M (LGM) showing the locations of the soybean aphid resistance genes R_ag1 and R_ag2. A. R_ag1 mapped in a Dowling x Loda mapping population; B. R_ag1 mapped in a Dowling x Williams 82 mapping population; C. Integrated map from Dowling x Loda and Dowling x Williams 82 mapping populations; D. R_ag2 mapped in a Jackson x Loda mapping population.

[0021] Figure 3. SSR markers showed co-segregation patterns with R_ag1. The ideal PCR amplified band pattern of a co-segregated marker would show A, B, B, B as corresponding to R (resistant parent Dowling, D), S (susceptible parent Loda, L), S (susceptible bulk 1 , S1), S (susceptible bulk 2, S2) phenotypes. Among the screened markers, four markers on soybean linkage group M showed potential co- segregation with R_ag1. Satt245 showed A, B, B, B pattern, Satt150, Satt220, and Satt323 showed A, B, B, H patterns.

DETAILED DESCRIPTION [0022] "Allele" is any of one or more alternative forms of a gene, all of which alleles relate to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes. The R_ag1 and R_ag2 genes may be allelic to each other. [0023] "Backcrossing" is a process through which a breeder repeatedly crosses hybrid progeny back to one of the parents (recurrent parent), for example, a first generation hybrid Fi with one of the parental genotypes of the Fi hybrid.

[0024] "Cultivar" and "variety" are used synonymously and mean a group of plants within a species (e.g., Glycine max) that share certain genetic traits that separate them from the typical form and from other possible varieties within that species. Soybean cultivars are inbred lines produced after several generations of self-pollination. Individuals within a soybean cultivar are homogeneous, nearly genetically identical, with most loci in the homozygous state.

[0025] "Gene" means a specific sequence of nucleotides in DNA that is located in the germplasm, usually on a chromosome, and that is the functional unit of inheritance controlling the transmission and expression of one or more traits by specifying the structure of a particular polypeptide or controlling the function of other genetic material. In the present instance, RAG genes for resistance to Aphis glycines (RAG) have been found on RAG loci flanked by markers Satt435 and Satt463. The RAG gene is referred to as R_ag1 when derived from or identical to the Dowling variety and as R_ag2 when derived from or identical to the Jackson variety. RAG genes may be isolated by one skilled in the art without undue experiments by means known to the art including PCR cloning utilizing the adjacent Satt435 and Satt463 primer sequences, or primer sequences from other markers flanking the gene as described herein, by positional cloning using BACs (bacterial artificial chromosomes), or other methods. See, e.g., Wu, et al., "A BAC and BIBAC-based Physical Map of the Soybean Genome" (2004) Genome Res. Feb;14(2):319-26, which describes the use of BACs in mapping the soybean genome. Contiguous BACs that have been found to be anchored to Satt435 and in which the R_ag1 gene may be found include B03I24^", B52J11*, B43I224^", H57B23, H03O08, B36M08*, H62M17, H75H01 , and E71J17. Information on these contiguous BACs is known to the art. Certain information is publicly available at the National Center for Biotechnology Information (NCBI) and GenBank web sites. The end sequence for H03O08 is set forth below: [0026] H03O08: AAGCTTCTAT CAAGTGGTAA TCAGAGCACA AGATCTTCAA GTAGGTGATC CTTAAACCTC CATTAATTTT TTGCTTTACC TTCTCTTCTA TTGTTGTTTC TTCATTTTTC TCCATGTATC TCCTCACATG TCTTGTGCTA AATGTTTTTA ACATGATTCT TTAGAGTTTC CACCGATTAA ACTTGCTATA GAAGCTAGAT TTGATTTTCT ATGGTTCAAA TTTCTTGTTC TTGTTCTTGA TCCATGAATT GTGTTGAGTT TAGGTTCCTT TGAGTTTTGT CTTGTTATTT TTTGTGGCTG AAACCTAAAC CATAAAATTC TTACAAAAAT ATTAAAGTAG AGGAAAACCT CAAAAATCTA GAGTGACTTG TTCACCTATT ATAGTTTTGT CATAGAAGTC ATGTCTAGTC ATGAAACTTG TCACATAAGA TTTCTTATGT TGTGCTGAAT TTTATTTTCT TGTTTCTTTG TCTAACTCAT TTGTTCATGA GTGTATGAAG TTATTTTAGC CTATTATTTG ATTGGAGTCA AATCTTTCAT GTTAATTAGT CCTTAACATG TTCATGCAAA ATTCTTAGAG AGTCTTTGAT TGTGAACCTT TTCTTGAACT TTTAGGTTTC CTTATGATTG TGTCTATTGT GAATTTAAGT TTTGGTGATT GAATTGCTGG TTGAAATGTT GATCCTAAGT GAATATTGAA CTCCTAAAAC TGTGGTAAAC AATCCTAGTG AGTTCAACAT ACATAGGAAG GTTGAAAGTA AGCCCAAGGC AATCAATATA GCATGCTTAA AAAAAAAATC GCTGGTGCTG GCAGCTTGGA CATACAAACT TGTAAAAATT ACTGAAAATT GGTTACTTCG AATTTTGAAC TGAATTTTTA CTTAATTTGC TAGA [SEQ ID NO:1]

[0027] "Germplasm" means the genetic material with its specific molecular and chemical makeup that comprises the physical foundation of the hereditary qualities of an organism. As used herein, germplasm includes seeds and living tissue from which new plants may be grown; or, another plant part, such as leaf, stem, pollen, or cells, that may be cultured into a whole plant. Germplasm resources provide sources of genetic traits used by plant breeders to improve commercial cultivars.

[0028] "Hybrid plant" means a plant offspring produced by crossing two genetically dissimilar parent plants.

[0029] "Inbred plant" means a member of an inbred plant strain that has been highly inbred so that all members of the strain are nearly genetically identical. [0030] "Introgression" means the entry or introduction by hybridization of a gene or trait locus from the genome of one plant into the genome of another plant that lacks such gene or trait locus.

[0031] "Molecular marker" is a term used to denote a nucleic acid or amino acid sequence that is sufficiently unique to characterize a specific locus on the genome. Examples include restriction fragment length polymorphisms (RFLPs) and single sequence repeats (SSRs). RFLP markers occur because any sequence change in DNA, including a single base change, insertion, deletion or inversion, can result in loss (or gain) of a restriction endonuclease recognition site. The size and number of fragments generated by one such enzyme is therefore altered. A probe that hybridizes specifically to DNA in the region of such an alteration can be used to rapidly and specifically identify a region of DNA that displays allelic variation between two plant varieties. SSR markers occur where a short sequence displays allelic variation in the number of repeats of that sequence. Sequences flanking the repeated sequence can serve as polymerase chain reaction (PCR) primers. Depending on the number of repeats at a given allele of the locus, the length of the DNA segment generated by PCR will be different in different alleles. The differences in PCR-generated fragment size can be detected by gel electrophoresis. Other types of molecular markers are known. All are used to define a specific locus on the soybean genome. Large numbers of these have been mapped. Each marker is therefore an indicator of a specific segment of DNA, having a unique nucleotide sequence. The map positions provide a measure of the relative positions of particular markers with respect to one another. When a trait is stated to be linked to a given marker it will be understood that the actual DNA segment whose sequence affects the trait generally co-segregates with the marker. More precise and definite localization of a trait can be obtained if markers are identified on both sides of the trait. By measuring the appearance of the marker(s) in progeny of crosses, the existence of the trait can be detected by relatively simple molecular tests without actually evaluating the appearance of the trait itself, which can be difficult and time-consuming, requiring growing up of plants to a stage where the trait can be expressed. [0032] Another type of molecular marker is the random amplified polymorphic DNA (RAPD) marker. Chance pairs of sites complementary to single octa- or decanucleotides may exist in the correct orientation and close enough to one another for PCR amplification. With some randomly chosen decanucleotides no sequences are amplified. With others, the same length products are generated from DNAs of different individuals. With still others, patterns of bands are not the same for every individual in a population. The variable bands are commonly called random amplified polymorphic DNA (RAPD) bands.

[0033] Another type of molecular marker is the target region amplification polymorphism (TRAP) marker. The TRAP technique employs one fixed primer of known sequence in combination with a random primer to amplify genomic fragments.

[0034] A further type of molecular marker is the single nucleotide polymorphism (SNP) marker, in which DNA sequence variations that occur when a single nucleotide (A₁T, C, or G) in the genome sequence is altered are mapped to sites on the soybean genome.

[0035] Other molecular markers known to the art, as well as phenotypic traits may be used as markers in the methods of this invention.

[0036] "Linkage" is defined by classical genetics to describe the relationship of traits that co-segregate through a number of generations of crosses. Genetic recombination occurs with an assumed random frequency over the entire genome. Genetic maps are constructed by measuring the frequency of recombination between pairs of traits or markers. The closer the traits or markers lie to each other on the chromosome, the lower the frequency of recombination, the greater the degree of linkage. Traits or markers are considered herein to be linked if they generally co-segregate. A 1/100 probability of recombination per generation is defined as a map distance of 1.0 centiMorgan (1.OcM). Preferably markers useful for screening for the presence of Aphis glycines resistance (RAG) map to within 20 cM of the trait, and more preferably within 10 cM of the trait. [0037] A second marker that maps to within 20 cM of a first marker that co- segregates with the RAG trait and generally co-segregates with the RAG trait is considered equivalent to the first marker. Any marker that maps within 20 cM and more preferably 10 cM of the RAG trait belongs to the class of preferred markers for use in screening and selection of soybean germplasm having the RAG Aphis glycines resistance trait. A number of markers are known to the art to belong to linkage group M on which the RAG trait is found. A number of markers are proprietary markers known only to certain of those skilled in the art of soybean plant breeding. A proprietary marker mapping within 20 cM, and preferably within 1 OcM, of any publicly known marker specified herein is considered equivalent to that publicly-known marker.

[0038] "Linkage group" refers to traits or markers that generally co-segregate. A linkage group generally corresponds to a chromosomal region containing genetic material that encodes the traits or markers.

[0039] "Locus" means a chromosomal region where a polymorphic nucleic acid or trait determinant or gene is located.

[0040] "Polymorphism" means a change or difference between two related nucleic acids. A "nucleotide polymorphism" refers to a nucleotide that is different in one sequence when compared to a related sequence when the two nucleic acids are aligned for maximal correspondence. A "genetic nucleotide polymorphism" refers to a nucleotide that is different in one sequence when compared to a related sequence when the two nucleic acids are aligned for maximal correspondence, where the two nucleic acids are genetically related, i.e., homologous, for example, where the nucleic acids are isolated from different strains of a soybean plant, or from different alleles of a single strain, or the like.

[0041] "Marker assisted selection" means the process of selecting a desired trait or desired traits in a plant or plants by detecting one or more nucleic acids from the plant, where the nucleic acid is linked to the desired trait. [0042] "Plant" means plant cells, plant protoplast, plant cell or tissue culture from which soybean plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants, such as seeds, pods, flowers, cotyledons, leaves, stems, buds, roots, root tips and the like.

[0043] "Probe" means an oligonucleotide or short fragment of DNA designed to be sufficiently complementary to a sequence in a denatured nucleic acid to be probed and to be bound under selected stringency conditions.

[0044]

resistance" or "R_ag2-derived resistance" means resistance in a soybean germplasm to Aphis glycines that is provided by the heterozygous or homozygous expression of the R_ag1 or R_ag2 gene within the RAG locus mapped between the SSR markers Satt435 and Satt463. "RAG-derived resistance" means Aphis glycines resistance conferred by a RAG gene on a RAG locus, the use of which is enabled by the disclosure herein.

[0045] "RAG phenotype" means resistance to Aphis glycines by soybean germpiasm, as demonstrated by resistance to Aphis glycines after inoculation with same according to the methods described herein.

[0046] "RAG soybean plant" means a plant having resistance to Aphis glycines that is derived from the presence and expression of at least one RAG gene, or that is shown to have a RAG gene at the RAG locus described herein.

[0047] "Self-crossing or self-pollination" is a process through which a breeder crosses hybrid progeny with itself, for example, a second generation hybrid F₂ with itself to yield progeny designated F₂:₃.

[0048] As used herein, the terms "segregate," "segregants," "co-segregate," "hybrid," "crossing," and "selfing" refer to their conventional meanings as understood in the art (see, for instance, Briggs, F. N. and Knowles, P. F. and, Introduction to Plant Breeding (Reinhold Publication Corp., New York, N.Y., 1967). [0049] Markers that "flank" the RAG genes are markers that occur one to either side of a RAG gene. Flanking marker DNA sequences may be part of the gene or may be separate from the gene.

[0050] The method for determining the presence or absence of a RAG gene, which confers resistance to the soybean aphid Aphis glycines in soybean germplasm, comprises analyzing genomic DNA from a soybean germplasm for the presence of at least one molecular marker, wherein at least one molecular marker is linked to the RAG trait locus, and wherein the RAG trait locus preferably maps to soybean major linkage group M and is associated with resistance to the soybean aphid Aphis glycines. The term "is associated with" in this context means that the RAG locus containing the RAG gene has been found, using marker-assisted analysis, to be present in soybean plants showing resistance to Aphis glycines in live aphid bioassays as described herein.

[0051] The R_ag1 gene occurs in the following varieties CNS (PI548445), and Dowling (PI548663), among others. The R_ag2 gene occurs in the following varieties: Jackson (PI548657), and Palmetto (PI548480), among others.

[0052] Other sources of A. glycines resistance include the G. max varieties: Moyashimame (PI87059), Sato (PI548409), Showa No. 1-4 (PI88508), Sugao Zarai (PI200538), T26OH (PI548237), PI71506, and PI230977 of G. max, and G3, JS1 , L4, S12 Taichung 38 (PI518282 and Taichung 37 (PI518281), of G. soja, and progeny of these varieties.

[0053] Table 1 lists Glycine max. varieties that are sources of resistance to the soybean aphid. Progeny of these varieties also containing a RAG gene are also sources of resistance to the soybean aphid. TABLE 1 SOURCES OF RESISTANCE TO SOYBEAN APHID

[0054] The following G. soja varieties are also sources of A. glycines resistance: PI441008, PI573059, and PI573071 , and progeny of these varieties.

[0055] Any one of the foregoing varieties or their progeny bearing a RAG gene may be used in the methods of this invention, and any combination thereof is considered to be a class of varieties useful in the methods of this invention.

[0056] Preferably a marker used to determine the presence or absence of a RAG gene is Satt435, Satt463, Satt245, S04309, S01623, or DOP_H14, or a marker that maps to within at least about 10 to about 20 cM of any of said markers.

Any marker assigned to soybean linkage group M may be useful for this purpose. Exemplary markers of linkage group M include Sat_389, Satt404, Sat_391 , GMSC514, Satt636, Satt590, GM177, GM175, Satt201 , Satt150, Sat_316, A351J , Mng339_1 , Ts, S01256, S02020, Satt567, Satt540, Bng222_1 , RGA_2b, RGA5b, GM260, S04309, Satt435, R079J , A060-2, DOPJH14, GM260, A131J , Sat_244, S01623, Satt463, Sat_253, S03544, Satt245, GM284, A946_2, GM256, GMS057, Satt220, A584_3, Satt626, OP_N04, Satt323, Sat_258, Satt702, L204_4, GMS003b, Satt536, Sat_003, OM11J 100, Sat_148, Bng179_2, K417_3, Sat_226, Satt175, K024_1 , A226_1 , GM230, ACCAGC315, Satt494, B157_2, A715J , Sct_147, Sat_256, Satt677, Sat_288, Satt655, Satt680, AF186183, BE823543, Satt306, A458_4, Satt728, Sat_422, E043_1 , Satt697, Mng186_1 , GM163, K070J , ACJ , BLT025_2, Cr326_3, Satt551 , M121J , Satt551 , Sat_131 , Satt250, Satt618, Satt210, Satt346, K227J , Sat_147, A064_1 , GM141 , GM209b, GM035a, A504_1 , Sat_276, Satt308, Satt336, Mng381_1 , Sat359, Sat_330, and A504.1.

[0057] Updated information regarding markers assigned to soybean linkage group M may be found on the USDA's Soybase website. Table 2 provides current information on the genbank location and allele size of markers useful in this invention. Table 3 provides upper and lower primer sequences. TABLE 2 - MARKERS

TABLE 3 - MARKER SEQUENCES

[0058] The sequence of the RAPD marker DOP_H14 is: 5' to 3': ACCAGGTTGG [SEQ ID NOM 04].

[0059] Table 4 provides information on additional SNP markers that are useful in practicing the present invention, showing their relative locations with respect to the markers described in Tables 2 and 3. [0060] TABLE 4 - SNP MARKERS

[0061] S01256 is available through: Genome Systems, Inc. 4633 World Parkway Circle St. Louis, Missouri. The sequence of S01256 and equivalent markers is taken from the 3' end of the following sequence: TATCATTATA TTGCAGGCTA CNNAAATTTC CAGTNNTAAT ACAGTATAAT TAAGCAGAGT GTGGTATCTA CAAAATCTCA ATCCAAACAC ATAATTACAA AACTCTAGAA CAGCAGAACA CATATAGCAT TTGATTTGAA GTATTCATTC ACTAATTGAT TAGCCTTAGA AATTCAAATG ATATAATCTG ACCACTCAGA GATAAAGGAA GTATGGTCCA TGGACTCCCC AGGAACATCC TCGTGCTTAG AGGGCTTCTC CTTCCCACCA ACCAACCTGG CTGGGTTCCC AACAGCTGTT GTCTGTGGTG GCACATCGAT TAAAACCACC GAGCCAGCAC AACCTTTGC ACCTTCCCCG ATCTTAATAT TCCCCAGAAT GGTAGCACCG GCACCAATAA GCACCCCATC CCCAATCTTG GGATGCCGGT CCCCACCAAC TTGCCAGTC CCACCCAGCG TAACGTGGTG CAGGATCGAC ACATTGTTCC GATCACTGC CGTCTCCCCC ACCACCACCC CGGTGGCATG GTCGAACAGA TCCCCTTCC CGATCCTCGC CGCAGGGTGA ATGTCCACCG CGAACACATC GCGATGCGA GAGTGCAGTG CNAAAGCCAA TGGCTGCCGC GATTGNCGNC CAACAGATG CGCCACACGG TGCGCCTGCA AAATCACAAT CACACACAAC TAATCCTAAG ATTCAATAAT CAAAAAAGAG TNNACTNNNC ATACACTGTC ATCNCNNNTA TAGTCATGTT TCATNNNAAT CTNGNNNNAC AATGCATATA AATTAAACTC AAT [SEQ ID NO:105]

[0062] S02020 is available through: Genome Systems, Inc. 4633 World Parkway Circle St. Louis, Missouri. The sequence of S02020 and equivalent markers is taken from the 3' end of the following sequence: AAAGNNAACA TTTTTGTTTA TATGACNNNA ACAAACTGCA AAGAAAAATT GTTAAAAACC AGAAGCAATT TAGGTGATCA CAAATACCAC ATGCTTACAC CTTCCAGTGA CAAGTACAGT ATGTTGTGGC ACCAGCCGTT TCAGTTGATG CAAACTTGCT TCGTGCCAAA ATTCTAACAA CACAACTACC TAAGCTATCA AACAAGAGAA GCCCTTTTGT CCTTTGGTCG ACCTATCAAA GGTCATCAGA TCACACTAGT CCTACCCTTT TAAGAAAACC TACTATCAAC AGTCATATGT ATCTCATGAA AAGCACATAA AAACATGTCA CTTTGCCTCT TCACCATCTC CACTGTTATG AGCAGCCGCG GAGCTGCCTT GGCCGTCTCC ACCAGCTGTT CCAGCCTCAG AGGCATCTTG CTTGCTTCCA CCACGTGCAT CGTTTGGACC AGTAGCCGAA GGTGGACCAC CGCTGTTTCC CCTCCAAGA GCAGCCTCAC TGTGCATTGG ATGCATGCCA TTATTTATAT CTCCAGGTCT AAGTCCCATT TGACCTTGGA TGGCCTGCTG GTGTAGCTGC TGTTGTTGTT CCTGCATTTG ATGTGGATTG CCAAATTGCA ATGGCATTTT CTGGGGGAAC ANNCCTTGCT GCTGCTGNNN NATTGCTGCT GCAGCNNNNT GNNNATNNNN NATATANNNN NC [SEQ ID NO:106]

[0063] S04309 is available through: Genome Systems, Inc. 4633 World Parkway Circle St. Louis, Missouri. The sequence of S04309 and equivalent markers is taken from the 3' end of the following sequence: TATCATTATA TTGCAGGCTA CNNAAATTTC CAGTNNTAAT ACAGTATAAT TAAGCAGAGT GTGGTATCTA CAAAATCTCA ATCCAAACAC ATAATTACAA AACTCTAGAA CAGCAGAACA CATATAGCAT TTGATTTGAA GTATTCATTC ACTAATTGAT TAGCCTTAGA AATTCAAATG ATATAATCTG ACCACTCAGA GATAAAGGAA GTATGGTCCA TGGACTCCCC AGGAACATCC TCGTGCTTAG AGGGCTTCTC CTTCCCACCA ACCAACCTGG CTGGGTTCCC AACAGCTGTT GTCTGTGGTG GCACATCGAT TAAAACCACC GAGCCAGCAC CAACCTTTGC ACCTTCCCCG ATCTTAATAT TCCCCAGAAT GGTAGCACCG GCACCAATAA GCACCCCATC CCCAATCTTG GGATGCCGGT CCCCACCAAC CTTGCCAGTC CACCCAGCG TAACGTGGTG CAGGATCGAC ACATTGTTCC CGATCACTGC CGTCTCCCCC ACCACCACCC CGGTGGCATG GTCGAACAGA ATCCCCTTCC GATCCTCGC CGCAGGGTGA ATGTCCACCG CGAACACATC AGCGATGCGA AGTGCAGTG CNAAAGCCAA TGGCTGCCGC GATTGNCGNC ACAACAGATG GCCACACGG TGCGCCTGCA AAATCACAAT CACACACAAC TAATCCTAAG ATTCAATAAT CAAAAAAGAG TNNACTNNNC ATACACTGTC ATCNCNNNTA TAGTCATGTT TCATNNNAAT CTNGNNNNAC AATGCATATA AATTAAACTC AAT [SEQ ID NO:107]

[0064] S01623 is available through: Genome Systems, Inc. 4633 World Parkway Circle St. Louis, Missouri. The sequence of S01623 and equivalent markers is taken from the 3' end of the following sequence: AAGACANNNN CGTTACATAA TCCTCACATA TAGTCATCCA ATCAGAACTG AATAGGAAAA AAAAATACAC AATATTAATG AAATTTAATT TATCATCTGC ATGTTTGGAT AAGCGTCAAA GGTAAACCTA CTATTAGTAG CTTTCTTGTC TTTCCTTCAA TTTGACGTGA TTTTAGTTTG AGACGTGCAT GTATAAAGTG GATCCAAACA CACTATTATG GTATGCAGAG TGAAGTAAAA ACTTAAAAAT CAGAGCAGCG ACCATTGCGT TCCCAGTCAC CATACCTAGT GGGCTCAGGC CCTTGGGTC CACCAATCTC ACCTGTTTCT TTGTTAATAC TGTCACCATC TTCGTGGTCT TCTTCGGGCT CATGGCTTTG TTTGTTCTCA TCATGGAGAG ATTCTTGAGG TGGTGTCTGT GCTTGTTCCC TGAGNGGGTT TTCGTGTTGT GGCTGAGTTG AAGAGCAGNN GAGCCGTGTC ACTGTGTTGG AAACAAAATG GTAAACTGC TCGGATTTGG TGCGGTGANN NNCNNTGTTG GCTACACAAG CAGTGAGCG AGGGAANNNG GTGGTCATTG TTGTTTGTTA ATGATGTAAG GCAGATGATC AGAAANNAGA AAACTCGTAN CNNNACGAAC AAAACCCTGA AATGGTTTAA AGCTNNNCCT TGGATTTTGA TTCTTGTTGC TGCGCGTTNG NNTGC [SEQ ID NO:108]

[0065] Markers that map closer to the RAG locus are preferred over markers that map farther from the RAG locus for use in this invention. A more preferred set of markers includes: Satt150, Sat_316, A351J , Mng339_1 , Ts, S01256, S02020, Satt567, Satt540, Bng222_1 , RGA_2b, RGA5b, GM260, S04309, Satt435, R079_1 , A060-2, DOP_H14, GM260, A131J , Sat_244, S01623, Satt463, Sat_253, S03544, Satt245, GM284, A946_2, GM256, GMS057, Satt220, A584_3, Satt626, OP_N04, Satt323, Sat_258, Satt702, L204_4, GMS003b, Satt536, Sat_003, OM11J 100, Sat_148, Bng179_2, K417_3, Sat_226, Satt175, K024J , A226J , GM230, ACCAGC315, Satt494, B157_2, A715_1 , and Sct_147.

[0066] A most preferred set of markers from which to choose at least one marker for use in this invention includes Satt435, Satt463, Satt245, S04309, S01623, and DOP_H14.

[0067] The markers may be any type of mapped molecular marker or phenotypic trait known to the art, including restriction fragment length polymorphism (RFLP) markers, target region amplification polymorphism (TRAP) markers, random amplified polymorphic (RAPD) markers, single sequence repeat (SSR) markers, single nucleotide polymorphism (SNP) markers, and isozyme markers.

[0068] In one embodiment of the invention, markers flanking the RAG locus are used in the marker-assisted selection processes of this invention. The genomic DNA of soybean germplasm is preferably tested for the presence of at least two of the foregoing molecular markers, one on each side of the RAG locus. Most preferably, the two markers are Satt435 and Satt463. Markers that map close to Satt435 and Satt463 can also be used, provided they fall to either side of the RAG locus. Preferably, one of said at least two molecular markers is within at least about 10 to about 20 cM of Satt435 and another of said at least two molecular markers is within at least about 10 to about 20 cM of Satt463, and to ensure that the markers used flank the RAG locus, one of said at least two molecular markers within at least about 10 to about 20 cM of Satt435 should be farther than that distance from Satt463, and another of said at least two molecular markers within at least about 10 to about 20 cM of Satt463 should be farther than that distance from Satt435.

[0069] The method of this invention for reliably and predictably introgressing soybean Aphis glycines resistance into non-resistant soybean germplasm or less resistant soybean germplasm comprises: providing a first soybean germplasm that has RAG-gene-derived resistance to Aphis glycines; providing a second soybean germplasm that lacks RAG-gene-derived resistance to Aphis glycines; crossing the first soybean germplasm with the second soybean germplasm to provide progeny soybean germplasm; screening said progeny germplasm to determine the presence of RAG-gene-derived resistance to Aphis glycines; and selecting progeny that tests positive for the presence of RAG-gene-derived resistance to Aphis glycines as being soybean germplasm into which germplasm having RAG-gene-derived resistance to Aphis glycines has been introgressed.

[0070] Preferably, the screening and selection are performed by using marker- assisted selection using a marker on major linkage group M as described above.

[0071] The screening and selection may also be performed by exposing plants containing said progeny germplasm to aphids of the species Aphis glycines in a live aphid assay and selecting those plants showing resistance to aphids as containing soybean germplasm into which germplasm having RAG-gene-derived resistance to Aphis glycines has been introgressed. The live aphid assay may be any such assay known to the art, e.g., as described in Hill, C. B., et al., "Resistance to the soybean aphid in soybean germplasm" (2004) Crop Science 44:98-106, Hill, CB. , et al., "Resistance of Glycine species and various cultivated legumes to the soybean aphid (Homoptera: Aphididae)" (2004) J. Economic Entomology 97(3)1071-1077, or "Li, Y. et al., "Effect of three resistant soybean genotypes on the Tecunαny, mortality, and maturation of soybean aphid (Homoptera: Aphididae)" (2004) J. Economic Entomology 97(3): 1106-1111 , or as described in the Examples hereof. A preferred method includes placing aphid-infested plant parts on vegetative cotyledon (VC) stage plants and rating aphid population and plant damage weekly. As described herein, a 0-5 rating scale in which 0 = no aphids present, 1 = a few solitary and transient aphids present, 2 = small scattered colonies, 3 = dense colonies, 4 = dense colonies with plant damage, and 5 = dense colonies with severe plant damage, may be used.

[0072] The screening and selection may also be done directly by hybridizing nucleic acid from plants containing progeny germplasm to a nucleic acid fragment comprising a RAG gene, and selecting those plants having germplasm that hybridizes to the nucleic acid fragment as having RAG-gene-derived resistance to Aphis glycines.

[0073] The method of this invention for breeding a soybean plant homozygous for an Aphis glycines resistance gene that is a cultivar adapted for conferring, in hybrid combination with a suitable second inbred, resistance to Aphis glycines, comprises selecting a first donor parental line possessing the desired Aphis glycines resistance, said first donor parental line comprising an Aphis glycines resistance gene that is located on major linkage group M; crossing the first donor parental line with a second parental line that is high yielding in hybrid combination to produce a segregating plant population of genetically heterogenous plants; screening the plants of the segregating plant population for the gene; selecting plants from the population having the gene; and breeding by self-crossing the plants containing the gene until a line is obtained that is homozygous for the locus containing the gene and adapted for conferring, in hybrid combination with a suitable second inbred, resistance to Aphis glycines.

[0074] The screening and selection are preferably performed by using marker- assisted selection as described above, but may also be performed by live aphid bioassay as described above, selecting those plants showing resistance to aphids as containing soybean germplasm having a RAG gene. The screening and selection may also be done by hybridizing nucleic acid from plants containing said progeny germplasm to a nucleic acid fragment comprising a RAG gene and selecting those plants whose germplasm hybridizes to the nucleic acid fragment as having the gene.

[0075] As the parental line having soybean aphid resistance, any line known to the art or disclosed herein, as described above, may be used.

[0076] Also included in this invention are soybean plants produced by any of the foregoing methods:

[0077] Isolated nucleic acid fragments comprising a nucleic acid sequence coding for soybean resistance to Aphis glycines, are also included in this invention. The nucleic acid fragment comprises at least a portion of nucleic acid belonging to linkage group M, and further comprises nucleotide sequences falling between molecular markers Satt435 and Satt463. It is capable of hybridizing under stringent conditions to nucleic acid of a soybean cultivar resistant to Aphis glycines.

[0078] Vectors comprising such nucleic acid fragments, expression products of such vectors expressed in a host compatible therewith, antibodies to the expression product (both polyclonal and monoclonal), and antisense nucleic acid to the nucleic acid fragment are also included within this invention.

[0079] This invention also includes soybean plants having resistance to Aphis glycines comprising a RAG gene and produced by introgression of DNA containing the gene into a soybean germplasm lacking the gene in its genome, and progeny of said soybean plant.

[0080] Seed of a soybean germplasm produced by crossing a soybean variety having Aphis glycines resistance in its genome with a soybean variety lacking the RAG gene in its genome, and progeny thereof, is also included in this invention. Such seed, from BC3 or BC4 generations derived from crosses with aphid resistant Dowling x Loda F₂ plants using as recurrent parents other soybean lines adapted to Illinois, is on deposit with the American Type Culture Collection (ATCC) under accession numbers , , and

EXAMPLES Example 1. Genetic analysis identifying the resistance gene R_ag1 in Dowling [0081] Crosses were made between the ancestral soybean cultivar Dowling and two susceptible cultivars, Loda and Williams 82. The parents, F-i, and F₂ plants were tested in a choice test in the greenhouse using the methods described in Hill, C. B., et al., "Resistance to the soybean aphid in soybean germplasm" (2004) Crop Science 44:98-106. Three weeks after infestation, aphid colonization was visually rated using the following scale: 0 = no aphids present, 1 = few solitary and transient aphids present, 2 = small scattered non-established colonies, 3 = dense colonies, and 4 = dense colonies with plant damage. Plants were considered resistant with a rating of 0, 1 , or 2 and susceptible with a rating of 3 or 4. Fi plants were all resistant to the soybean aphid, indicating that resistance was dominant over susceptibility, χ² analyses on the segregation of resistance phenotypes of F₂ plants from different Fi plants (families) indicated that a single dominant gene, called R_ag1, pending approval by the Soybean Genetics Committee, conditioned resistance (Tables 5 and 6). Evaluation of the segregation of aphid resistance in F^.z families confirmed the monogenic dominant inheritance of resistance from Dowling (Table 8). TABLE 5

REACTIONS OF DOWLING x LODA F₂ PLANTS AND PARENTS 21 DAYS

AFTER INFESTATION BY THE SOYBEAN APHID

¹R (resistant) = 0, 1 , 2 aphid colonization rating; S (susceptible) = 3, 4 rating. ²One Loda plant had an aphid colonization rating of 2.

TABLE 6

REACTIONS OF DOWLING x WILLIAMS 82 F₂ PLANTS

AND PARENTS 21 DAYS AFTER INFESTATION BY THE SOYBEAN APHID

¹R (resistant) = 0, 1 , 2 aphid colonization rating; S (susceptible) = 3, 4 rating. Example 2. Genetic analysis identifying the resistance gene R_ag2 in Jackson [0082] Crosses were made between the ancestral soybean cultivar Jackson and Loda. The parents, F-i, and F₂ plants were tested in a choice test in the greenhouse using the methods described in Hill, C. B., et al., "Resistance to the soybean aphid in soybean germplasm" (2004) Crop Science 44:98-106. Three weeks after infestation, aphid colonization was visually rated using the following scale: 0 = no aphids present, 1 = few solitary and transient aphids present, 2 = small scattered colonies, 3 = dense colonies, and 4 = dense colonies with plant damage. Plants were considered resistant with a rating of 0, 1 , or 2 and susceptible with a rating of 3 or 4. Fi plants were all resistant to the soybean aphid, indicating that resistance was dominant over susceptibility, χ² analyses on the segregation of resistance phenotypes of F₂ plants from different F₁ plants (families) indicated that a single dominant gene, called R_ag2, pending approval by the Soybean Genetics Committee, conditioned resistance (Table 7). Evaluation of the segregation of aphid resistance in F_2:3 families indicated that the segregation of families did not fit a monogenic dominant inheritance model (Table 8). The unexpected F_2:3 family segregation ratio may have been due to differential seed production between resistant and susceptible F₂ plants. Progeny of F₂ plants that produced at least 12 seeds were evaluated so that number plants tested exceeded the minimum required (10 plants) to have high confidence (95%) in detecting double recessive susceptible plants in segregating families with a monogenic dominant gene model. About 80% of the resistant F₂ plants produced at least 12 seeds, whereas about 17% of the susceptible F₂ plants produced 12 seeds or more. TABLE7 REACTIONS OFJACKSONXLODAF₂ PLANTSAND PARENTS 21 DAYS AFTERINFESTATION BYTHE SOYBEANAPHID.

R (resistant) = 0, 1 , 2 aphid colonization rating; S (susceptible) = 3, 4 rating TABLE 8 REACTIONS OF DOWLING X LODA, DOWLING x WILLIAMS 82, AND JACKSON x LODA F_2:3 FAMILIES AND 21 DAYS AFTER INFESTATION BY THE SOYBEAN APHID

¹12 seeds of each F₂ plant were sown. ²R = all plants in an F_2:3 family were resistant, H = plants in a family segregated for resistance, S = all plants in a family were susceptible.

Example 3. Molecular markers linked to R_ag1 [0083] A soybean F₂ population developed from a cross between Dowling x Loda was used for mapping the location of R_ag1. A total of 90 F₂ individuals and the two parents were included in the mapping work. The phenotypic data (aphid colonization on F₂ plants) was scored as described above in the genetic analysis.

[0084] For genotypic data, DNA was isolated from individual plants and polymerase chain reaction (PCR) was carried out using simple sequence repeat (SSR) markers developed by Dr. Perry Cregan, USDA-ARS (See Table 2). The PCR products were evaluated on gels as previously described in Wang, D.J. et al., "A low-cost, high-throughput polyacrylamide gel electrophoresis system for genotyping with micro satellite DNA markers," (2003) Crop Science 43:1828-1832. Initial screening was done using the parents and two bulked DNA samples to identify polymorphic simple sequence repeat (SSR) markers. Each bulk consisted of pooled DNA samples from five susceptible F₂ individuals. A total of about 342 SSR markers were screened against the bulks to identify polymorphic markers potentially associated with aphid resistance. Markers showing strong association with R_ag1 were further screened using the entire mapping population to determine linkage relationships and map locations. Joinmap 3.0 was used to create a genetic map. As shown in Figure 1 , R_ag1 mapped to Linkage Group M where it is flanked by the SSR markers Satt435 and Satt463 that are 3cM and 6cM from the R_ag1 locus, respectively.

Example 4. Molecular markers linked to R_ag1 in Jackson [0085] A soybean F₂ population developed from a cross between Jackson and Loda was used for mapping the location of the resistance gene to Aphis glycines. A total of 92 F₂ individuals and the two parents were included in the mapping work.

[0086] The phenotypic data (aphid colonization on F₂ plants) was scored as described above in the genetic analysis.

[0087] For genotypic data, DNA isolation, PCR, and gel electrophoresis were done as described in Wang, D.J. et al., "A low-cost, high-throughput polyacrylamide gel electrophoresis system for genotyping with micro satellite DNA markers," (2003) Crop Science43: 1828-1832). Three SSR markers, Satt435, Satt463, and Satt245, which are mapped 3cM, 6cM, and 13cM from R_ag1 in Dowling (Figure 2), respectively, showed polymorphism between Jackson and Loda and are associated with aphid resistance in Jackson based on 14 F₂ individuals. These three markers were further screened using the entire mapping population to determine linkage relationships and map locations.

[0088] Joinmap 3.0 was used to create a genetic map. As shown in Figure 2, the Aphis glycines gene locus also mapped to Linkage Group M where the SSR marker Satt435 is 9cM away from the R_ag2 locus.

Example 5. Location of R_ag1 in the soybean genetic map

[0089] F_2:3 populations from the cross between Dowling and the two susceptible soybean cultivars, Loda and Williams 82, were used to map R_ag1 in Dowling using linked SSR markers Satt150, Satt540, Satt435, Satt463, Satt245, Satt220 and Satt323. See Tables 2 and 3. [0090] One hundred and forty nine F₂ plants and their F_2:3 families from Dowling x Loda were used for initial marker screening and initial mapping of R_ag1 in Dowling. One hundred and twenty one F_2:3 families from Dowling x Williams 82 were used to confirm the R_ag1 map location and to construct an integrated map for R_ag1 in Dowling.

[0091] In the integrated map from Dowling x Loda and Dowling x Williams 82 populations, R_ag1 was mapped to soybean linkage group M flanked by the SSR markers Satt435 and Satt463 4.0 cM and 8.0 cM from R_ag1, respectively (Tables 9 and 10; Figures 1 and 2).

Plant materials [0092] Three F_2:3 populations from the crosses "Dowling" (Pl 548663) x "Loda" (Pl 614088), Dowling x "Williams 82" (Pl 518671) and "Jackson" (Pl 548657) x Loda, and one F₂ population of Dowling x Palmetto (Pl 548480), were used in this study based on crosses made by Curt Hill. Dowling, Jackson and Palmetto are aphid resistant while Loda and Williams 82 are aphid susceptible. Palmetto was suggested as the origin of the resistance in Jackson because it is the only known resistant ancestor of Jackson (Hill, C. B., et al., "Resistance to the soybean aphid in soybean germplasm" (2004) Crop Science 44:98-106). One hundred and forty nine F₂ plants and their F₂.₃ families from Dowling x Loda were used for initial marker screening and initial mapping of R_ag1 in Dowling. One hundred and twenty one F_2:3 families from Dowling x Williams 82 were used to confirm the R_ag1 map location and to construct an integrated map for R_ag1 in Dowling. One hundred and forty F₂ plants and their F_2:3 families from Jackson x Loda were used to map R_ag2 in Jackson. Sixty-five F₂ plants from Dowling x Palmetto were used to test allelism indirectly between R_ag1 in Dowling and R_ag2 in Jackson. Dowling x Jackson crosses were not made because there are no known polymorphic markers known that could be used to distinguish F1 hybrids from selfs in crosses.

Aphid clone [0093] The aphid clone was collected from Urbana, Illinois by Dr. Les Domier (USDA-ARS and Department of Crop Sciences, University of Illinois, Urbana, IL 61801) and reared on the seedlings of soybean cultivar Williams 82 in a plant growth chamber at 22⁰C under continuous 200 μmol m-2 s-1 PAR irradiation.

Soybean aphid resistance phenotyping [0094] The parents, F₂ plants, and susceptible checks were screened for aphid resistance under semi-controlled conditions (22-25⁰C under continuous 24-h illumination (160-200 μmol m^"2 s^"1) in the greenhouse. In a randomized complete block design, seeds were grown in plastic multi-pot inserts within plastic trays without holes. One week later, soybean aphids were transferred from the infested Williams 82 cut stems and leaves to the young test seedlings. Each individual plant was evaluated for aphid score twice at 14 days and 21 days after infestation. Aphid score was rated as index based on aphid population density and plant damage: 0- 4, where 0 = no aphids observed, 1 = few number of aphids scattered on the plant, 2 = limited colonization of aphids observed, 3 = high aphid density and colonization, 4 = high aphid density and colonization plus leaf distortion and plant stunting. After the 21-day rating, insecticide (imidacloprid) was applied. After one week, leaf tissue from the F₂ plants was sampled for DNA extraction. All F₂ plants were transplanted to 5-inch diameter plastic pots and were grown in the greenhouse under a 12h photoperiod to produce F₃ seeds. In the progeny test, 10-12 F₃ seeds per F₂ family were evaluated for aphid resistance in a randomized complete block design with three replicates (four F₃ plants per F₂ family per replicate). F₂ genotypes (homozygous resistant, heterozygote, or homozygous susceptible) were inferred from the segregation of the F₃ plants.

DNA isolation, PCR reaction and gel electrophoresis [0095] Young trifoliolate leaves were harvested from the new growth of each individual plant after the aphids were killed. Soybean DNA was extracted from either an individual F₂ plant or pooled 10-12 F₃ plants, by using either the CTAB method (Keim, P. and Shoemaker, R. C, "Construction of a random recombinant DNA library that is primarily single copy sequence" (1988) Soybean Genet. Newslet. 15:147-148), or DNA quick extraction method (Bell-Johnson, B. et al., "Biotechnology approaches to improving resistance to SCN and SDS: methods for high throughput marker assisted selection" (1998) Soybean Genet. Newslet. 25:115-117). [0096] The PCR amplification was performed in a PTC-220 Thermalcycler manufactured by MJ Research (Waltham, MA). PCR reactions were done in 15 μl volumes with 50-250 ng of template DNA, 2 μM primer, 30 mM MgCI₂, 3 mM each dNTP, 2.5 unit of Taq polymerase, and 1 x PCR buffer. The PCR was performed with an initial denaturing at 94⁰C for 4 min, followed by 34 cycles of 25 s of denaturing at 94 °C, 25 s of annealing at 47 ⁰C, and 25 s of extension at 68 ⁰C, with a final 7-min extension at 72⁰C.

[0097] The gel electrophoresis was done using non-denaturing polyacrylamide gels as described before (Wang, DJ. et al., "A low-cost, high-throughput polyacrylamide gel electrophoresis system for genotyping with micro satellite DNA markers," (2003) Crop Science43:1828-1832). After electrophoresis the gels were photographed and the polymorphic bands were scored as described below.

SSR marker screening and bulk segregant analysis [0098] Soybean simple sequence repeat (SSR) markers developed by Dr. Cregan (Cregan, P. B., et al., "An Integrated Genetic Linkage Map of the Soybean Genome" (1999) Crop Science39:1464-1490) were used in this study. Bulk segregant analysis (Michelmore, R.W., et al., "Identification of markers linked to disease resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions using segregating populations" (1991)Proc. Natl. Acad. Sci. (USA) 88:9828-9832 in Dowling x Loda F₂ population was used to screen for aphid-resistance associated markers. Since at the time of screening, only F₂ individuals were available, the resistant F₂ plants could be either heterozygous or homozygous, therefore susceptible bulks were used to look for linkage. Two pools of DNA from five susceptible F₂ individuals each, bulk A and bulk B were prepared. DNA from resistant parent Dowling and susceptible parent Loda, along with the DNA pools bulk A and B were used to identify polymorphic SSR markers with potential association with aphid resistance. 342 SSR markers were tested for polymorphism between two parents Dowling and Loda, and the segregation patterns for the aphid resistance versus susceptibility. The polymorphic markers with putative linkage with R_ag1 were first identified by contrasting bulk segregant analysis, and then were further screened in the whole Dowling x Loda mapping population. [0099] The SSR markers that were determined to map close to R_ag1 in the Dowling x Loda mapping population were used to test the polymorphism between Dowling and Williams 82 and between Jackson and Loda. The polymorphic markers were then further screened in the entire population of Dowling x Williams 82 and Jackson x Loda. The data from Dowling x Loda and Dowling x Williams 82 was integrated together to map R_ag1 in Dowling, and the data from Jackson x Loda was used to map R_ag2 in Jackson.

[00100] The two SSR markers that flanked the R_ag1 gene in Dowling and R_ag2 in Jackson were found to be monomorphic in Palmetto also, the parent of Jackson.

[00101] Genetic mapping Joinmap 3.0 (Van Ooijen, J.W. and Voorrips, V.E., JoinMap 3.0, Software for the calculation of genetic linkage maps. Plant Research International, Wageningen, the Netherlands (2001)) was used for linkage analysis to create a genetic map using the Kosambi mapping function. A LOD of 3.0 was used as the threshold to group markers into linkage groups. Chi square (χ²) test at P = 0.05 was used to verify the segregation ratio of each locus in the F₂ population. The genotypes of the SSR markers were scored as either co-dominant (A=RR, H=Rr, B=rr) or dominant (D=A+H, B or A, C=B+H). Genotypes of the F₂ aphid population were scored as co- dominant (A, H, B) after confirmation with the F₃ progeny test. Those that had less than 10 F₃ seeds available from the F₂ plant were scored as dominant (D, B). All of the SSR markers and aphid resistance (R_ag1 and R_ag2) were set to the corresponding χ²-test classification as described in Joinmap 3.0, 1 :2:1 (A: H: B) or 3:1 (A+H+D: B=3:1) segregation ratio. TABLE 9 χ²-TEST OF THE SEGREGATION RATIO FOR R_AG1 AND THE LINKED SSR MARKERS IN THE F₂ POPULATION FROMTHE CROSS OF DOWLING x LODA.

TABLE 10 χ²-TEST OF THE SEGREGATION RATIO FOR R_AG1 AND THE LINKED SSR MARKERS IN THE F₂ POPULATION FROM THE CROSS OF DOWLING X WILLIAMS 82

Example 6. Location of R_ag2 in the soybean genetic map

[00102] One hundred and forty F₂ plants and their F_2:3 families from Jackson x Loda were used to map R_ag2 in Jackson using linked SSR markers (Tables 2 and 3).

[00103] R_ag2 was mapped to linkage group M flanked by markers Satt435 and Satt463 1.9 cM and 7.7 cM, respectively (Table 11 ; Figures 1 and 2). TABLE 11 χ²-TEST OF THE SEGREGATION RATIO FOR R_AG2 AND THE LINKED SSR MARKERS IN THE F₂ POPULATION FROM THE CROSS OF JACKSON x LODA

[00104] Although the foregoing invention has been described in detail for purposes of clarity and understanding, it will be clear to those skilled in the art that equivalent cultivars, markers, and methods may be practiced within the scope of the claims hereof.

Claims

1. A method for determining the presence or absence of a gene for resistance to Aphis glycines in soybean germplasm comprising:

analyzing genomic DNA from a soybean germplasm for the presence of at least one molecular marker, wherein the at least one molecular marker is linked to the RAG trait locus and wherein the RAG trait locus maps to soybean major linkage group M and is associated with resistance to the soybean aphid Aphis glycines.

2. The method of claim 1 wherein said gene is R_ag1 orR_ag2.

3. The method of claim 1 wherein said marker is selected from the group consisting of Satt435, Satt463, Satt245, S04309, S01623, DOPJH14, and markers that map to within at least about 20 cM of any of these markers.

4. The method of claim 1 wherein said marker is selected from the group consisting of Sat_389, Satt404, Sat_391 , GMSC514, Satt636, Satt590, GM177, GM175, Satt201 , Satt150, Sat_316, A351J , Mng339J , Ts, S01256, S02020, Satt567, Satt540, Bng222_1 , RGA_2b, RGA5b, GM260, S04309, Satt435, R079J , A060-2, DOPJH14, GM260, A131J , Sat_244, S01623, Satt463, Sat_253, S03544, Satt245, GM284, A946_2, GM256, GMS057, Satt220, A584_3, Satt626, OP_N04, Satt323, Sat_258, Satt702, L204_4, GMS003b, Satt536, Sat_003, OM11_1100, Sat_148, Bng179_2, K417_3, Sat_226, Satt175, K024J , A226_1 , GM230, ACCAGC315, Satt494, B157_2, A715_1 , Sct_147, Sat_256, Satt677, Sat_288, Satt655, Satt680, AF186183, BE823543, Satt306, A458_4, Satt728, Sat_422, E043_1 , Satt697, Mng186_1 , GM 163, K070J , ACJ , BLT025_2, Cr326_3, Satt551 , M121J , Satt551 , Sat_131 , Satt250, Satt618, Satt210, Satt346, K227J , SaM 47, A064_1 , GM141 , GM209b, GM035a, A504J , Sat_276, Satt308, Satt336, Mng381_1 , Sat359, Sat_330, and A504.1.

5. The method of claim 1 wherein said marker is selected from the group consisting of Satt150, Sat_316, A351J , Mng339_1 , Ts, S01256, S02020, Satt567, Satt540, Bng222_1 , RGA_2b, RGA5b, GM260, S04309, Satt435, R079J , A060-2, DOP_H14, GM260, A131J , Sat_244, S01623, Satt463, Sat_253, S03544, Satt245, GM284, A946_2, GM256, GMS057, Satt220, A584_3, Satt626, OP_N04, Satt323, Sat_258, Satt702, L204_4, GMS003b, Satt536, Sat_003, OM11J 100, Sat_148, Bng179_2, K417_3, Sat_226, Satt175, K024J , A226_1 , GM230, ACCAGC315, Satt494, B157_2, A715_1 , and Sct_147.

6. A method for reliably and predictably introgressing soybean Aphis glycines resistance into non-resistant soybean germplasm or less resistant soybean germplasm comprising:

providing a first soybean germplasm that has resistance to Aphis glycines;

providing a second soybean germplasm that lacks resistance to Aphis glycines;

crossing the first soybean germplasm with the second soybean germplasm to provide progeny soybean germplasm;

analyzing said progeny germplasm to determine the presence of resistance to Aphis glycines; and

selecting progeny that tests positive for the presence of resistance to Aphis glycines as being soybean germplasm into which germplasm having resistance to Aphis glycines has been introgressed.

7. The method of claim 6 wherein said analysis of said progeny germplasm is performed by analyzing genomic DNA from the germplasm for the presence of at least one molecular marker, wherein the at least one molecular marker is linked to the RAG trait locus and wherein the RAG trait locus maps to soybean major linkage group M and is associated with resistance to the soybean aphid Aphis glycines.

8. The method of claim 7 wherein said marker is selected from the group consisting of Satt435, Satt463, Satt245, S04309, S01623, DOPJH14, and markers that map to within at least about 20 cM of any of these markers.

9. The method of claim 7 wherein said marker is selected from the group consisting of Sat_389, Satt404, Sat_391 , GMSC514, Satt636, Satt590, GM177, GM175, Satt201 , Satt150, Sat_316, A351J , Mng339_1 , Ts, S01256, S02020, Satt567, Satt540, Bng222_1 , RGA_2b, RGA5b, GM260, S04309, Satt435, R079J , A060-2, DOPJH14, GM260, A131J , Sat_244, S01623, Satt463, Sat_253, S03544, Satt245, GM284, A946_2, GM256, GMS057, Satt220, A584_3, Satt626, OP_N04, Satt323, Sat_258, Satt702, L204_4, GMS003b, Satt536, Sat_003, OM11_1100, Sat_148, Bng179_2, K417_3, Sat_226, Satt175, K024J , A226J , GM230, ACCAGC315, Satt494, B157_2, A715_1 , Sct_147, Sat_256, Satt677, Sat_288, Satt655, Satt680, AF186183, BE823543, Satt306, A458_4, Satt728, Sat_422, E043J , Satt697, Mng186_1 , GM163, K070J , ACJ , BLT025_2, Cr326_3, Satt551 , M121J , Satt551 , Sat_131 , Satt250, Satt618, Satt210, Satt346, K227J , Sat_147, A064_1 , GM141 , GM209b, GM035a, A504J , Sat_276, Satt308, Satt336, Mng381_1 , Sat359, Sat_330, and A504.1.

10. The method of claim 7 wherein said marker is selected from the group consisting of Satt150, Sat_316, A351J , Mng339_1 , Ts, S01256, S02020, Satt567, Satt540, Bng222_1 , RGA_2b, RGA5b, GM260, S04309, Satt435, R079J , A060-2, DOPJH14, GM260, A131J , Sat_244, S01623, Satt463, Sat_253, S03544, Satt245, GM284, A946_2, GM256, GMS057, Satt220, A584_3, Satt626, OP_N04, Satt323, Sat_258, Satt702, L204_4, GMS003b, Satt536, Sat_003, OM11J 100, Sat_148, Bng179_2, K417_3, Sat_226, Satt175, K024J , A226_1 , GM230, ACCAGC315, Satt494, B157_2, A715_1 , and Sct_147.

11. The method of claim 6 wherein said analysis of said progeny germplasm is performed by exposing plants containing said progeny germplasm to aphids of the species Aphis glycines in a live aphid assay and selecting those plants showing resistance to aphids.

12. The method of claim 6 wherein said soybean germplasm that has resistance to Aphis glycines is germplasm of a variety having a Plant Introductions (Pl) number selected from the group consisting of 71506, 87059, 88508, 200538, 230977, 417084A, 437696, 499955, 507298, 508294, 518726, 548237, 548409, 548445, 548480, 548657, 548663, 567391 , 567541 B, 567543C, 567597C, 567598B, 587552, 587553A, 587559B, 587617, 587656, 587663, 587664B, 587666, 587668A, 587669, 587674A, 587677, 587682A, 587684A, 587685, 587686A, 587687A, 587693, 587700A, 587702, 587717, 587723A, 587732, 587759, 587763, 587775, 587800, 587816, 587824, 587840, 587844C, 587861 , 587863B, 587870, 587871 , 587873, 587876, 587877A, 587891A, 587897, 587899, 587905, 587972, 588000, 588040, 594421 , 594425, 594426A, 594426B, 594427A, 594431 , 594499, 594503, 594514, 594554, 594557B, 594560B, 594573, 594586A, 594592, 594595, 594666B, 594703, 594707, 594711 B, 594751 A, 594822, 594864, 594868, 594879, 603521 , 603530A, 603538A, 603640, 603644, 603650, 605771 , 605823, 605855, and 605902, and progeny of these varieties having Aphis glycines resistance.

13. The method of claim 6 wherein said soybean germplasm that has resistance to Aphis glycines is germplasm of a variety selected from the group consisting of CNS (PI548445) and Dowling (PI548663), and progeny of these varieties having Aphis glycines resistance.

14. The method of claim 6 wherein said soybean germplasm that has resistance to Aphis glycines is germplasm of a variety selected from the group consisting of Jackson (PI5478657) and Palmetto (PI548480), and progeny of these varieties having Aphis glycines resistance.

15. The method of claim 6 wherein said soybean germplasm that has resistance to Aphis glycines is germplasm of a variety selected from the group consisting of Moyashimame (PI87059), Sato (PI548409), Showa No. 1-4 (PI88508), ), Sugao Zarai (PI200538), T26OH (PI200538), PI71506, and PI230977 of G. max, and G3, JS1 , L4, S12 Taichung 38 (PI518282 and Taichung 37 (PI518281),of G. soja. and progeny of these varieties having Aphis glycines resistance.

16. A soybean plant made by the method of claim 6.

17. The method of claim 6 for the production of an inbred soybean cultivar adapted for conferring, in hybrid combination with a suitable second inbred, resistance to Aphis glycines, wherein said second soybean germplasm that lacks resistance to Aphis glycines is high yielding in hybrid combination with said first soybean germplasm that has resistance to Aphis glycines; comprising the further step of self-crossing the plants that tested positive for the presence of Aphis glycines resistance until a line is obtained that is homozygous for resistance to Aphis glycines and adapted for conferring, in hybrid combination with a suitable second inbred, resistance to Aphis glycines.

18. A soybean plant produced by the method of claim 17.

19. An isolated nucleic acid fragment comprising a nucleic acid sequence coding for soybean resistance to Aphis glycines, said nucleic acid fragment comprising at least a portion of nucleic acid belonging to linkage group M, said nucleic acid fragment further comprising nucleotide sequences falling between molecular markers Satt435 and Satt463, and said nucleotide fragment further being capable of hybridizing under stringent conditions to nucleic acid of a soybean cultivar resistant to Aphis glycines.

20. The nucleic acid fragment of claim 19 comprising an Aphis glycines resistance gene selected from the group consisting of R_ag1 and R_ag2.

21. The nucleic acid fragment of claim 20 derived from a cultivar selected from the group consisting of CNS (PI548445), Dowling (PI548663), Jackson (PI548657), Palmetto (PI548480) and progeny of these varieties having Aphis glycines resistance.

22. The nucleic acid fragment of claim 21 derived from PI071506, and progeny of this variety having A. glycines resistance.

23. A vector comprising the nucleic acid fragment of claim 20.

24. An expression product of a vector of claim 23 expressed in a host compatible with said vector.

25. An antibody to the expression product of claim 24.

26. Antisense nucleic acid to the nucleic acid fragment of claim 20.

27. Seed of a soybean germplasm produced by crossing a soybean variety having Aphis glycines resistance in its genome with a soybean variety lacking such resistance, said seed being selected from the group consisting of seed of soybean germplasms that are developed by crossing Dowling with Loda, and backcrossing the F₂ generation thereof for three or four generations, said seed being deposited as ATCC accession numbers , , and , respectively, and progeny therefrom.